WO2002073164A1 - Method for analyzing substance - Google Patents

Method for analyzing substance Download PDF

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Publication number
WO2002073164A1
WO2002073164A1 PCT/JP2001/001854 JP0101854W WO02073164A1 WO 2002073164 A1 WO2002073164 A1 WO 2002073164A1 JP 0101854 W JP0101854 W JP 0101854W WO 02073164 A1 WO02073164 A1 WO 02073164A1
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Prior art keywords
fine particles
lyophobic
substance
analysis method
particles
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PCT/JP2001/001854
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French (fr)
Japanese (ja)
Inventor
Takao Fukuoka
Keitaro Nakamura
Yasushige Mori
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Takao Fukuoka
Keitaro Nakamura
Yasushige Mori
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Application filed by Takao Fukuoka, Keitaro Nakamura, Yasushige Mori filed Critical Takao Fukuoka
Priority to US10/469,932 priority Critical patent/US7198957B2/en
Priority to PCT/JP2001/001854 priority patent/WO2002073164A1/en
Priority to CNA200610114882XA priority patent/CN1982868A/en
Priority to CNB018230113A priority patent/CN1278115C/en
Priority to EP01912222.5A priority patent/EP1376098B1/en
Priority to KR1020037011728A priority patent/KR100634663B1/en
Priority to JP2002572378A priority patent/JP4772273B2/en
Publication of WO2002073164A1 publication Critical patent/WO2002073164A1/en
Priority to US11/716,553 priority patent/US7374873B2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
    • G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light

Definitions

  • the present invention relates to all analytical methods utilizing the surface enhancement effect in vibrational spectroscopy.
  • it relates to the life science field that requires analysis of biological components such as functional analysis of protein in aqueous solution, and the global and environmental fields that require analysis of harmful substances in the environment.
  • Biofunctional substances have activity in living organisms, which use water as a medium. Therefore, elucidation of its physicochemical properties also needs to be performed in aqueous solution.
  • Raman spectroscopy a type of vibrational spectroscopy, is a useful analytical method for examining the structure of bio-functional substances and their interaction with substrate molecules even in water.
  • the surface enhancing effect is remarkably exhibited when the metal fine particles are used in an aggregated state. Measurement methods using the surface enhancement effect are also important as biotechnology research tools (K. Kneipp, H. Kneipp, I. Itzkan, RR Dasari, and MS Fe Id, Biomedical Applications of Lasers, 77 (7 ), 915-924 (1999); Surface-enhanced Raman scattering: A new tool for biomedical spectroscopy).
  • Examples of the mode of using metal fine particles as a substrate for the surface enhancement effect include a colloid of metal fine particles, a film in which metal fine particles are deposited in an island shape on the surface, a glass matrix in which metal fine particles are dispersed inside by a sol-gel method, A polymer matrix in which metal fine particles are dispersed has been reported so far. Further, the present inventors also disclose in Japanese Patent Application Laid-Open No. Hei 11-116209 that a noble metal fine particle is reduced by a reduction reaction in a dispersion liquid in which plate-like fine particles such as a swellable layered silicate are dispersed. A technique for obtaining a stable dispersion of noble metal fine particles by producing the same has been disclosed.
  • colloids of nano-noble metal particles in aqueous solution are said to be the most practical in practice.
  • the reasons are: 1) Fine particles can be synthesized by a liquid phase method, and handling is easy. 2) Applicable to continuous flow analysis system. 3) Particle size and shape can be controlled. 4) The surface area can be easily defined. 5) Can change form for theoretical analysis. (M. Kerker, DS Wang, H. Chew. 0. Si iman, and LA Bumm, "Surface Enhanced Raman Scattering", ed. By RK Chang and TE Furtak, (Plenum Publishing, NY, 1982), pp. 109-128; Enhanced Raman scattering by molecules adsorbed at the surface of colloidal particles).
  • the dispersion state has been controlled by the following methods: 1) adding a stabilizer in the liquid phase, and 2) depositing (coating) on the solid phase because the metal fine particles are lyophobic fine particles. 3) Incorporation of glass, polymer, etc. into the matrix as described above, 4) Swellable layered silicate as fine metal particles as disclosed in JP-A-11-161209. It has been proposed to coexist.
  • the stabilizers used in the liquid phase include surfactants such as sodium dodecyl sulfate, polyvinyl alcohol, polyvinyl pyridine, polyethylene glycol, N-vinyl pyrrolidone, sera albumin, and var.
  • surfactants such as sodium dodecyl sulfate, polyvinyl alcohol, polyvinyl pyridine, polyethylene glycol, N-vinyl pyrrolidone, sera albumin, and var.
  • Protective colloids such as gelatin and gelatin are known.
  • Japanese Patent Application Laid-Open No. 09-070527 “Method for Preventing Colloid Aggregation" discloses a stabilizing effect of a buffer such as trishydroxymethylaminoaminomethane.
  • the synthetic smectite is modified with one or more substances selected from the group consisting of a ligand compound, an antibody, an antigen, an enzyme, an enzyme substrate, a nucleic acid, and a nucleic acid complement, and It can have the function of recognizing or orienting substances.
  • the surface enhancement effect is significantly reduced when the distance from the surface of the lyophobic fine particles is increased, so that only the recognized substance or some of the functional groups due to the orientation have an effect on the surface enhancement effect. It can be used for selective substance measurement.
  • Fig. 1 shows the absorption spectrum of the sol containing the fine gold particles and synthetic smectite
  • Fig. 2 shows the light absorption spectrum of the sol 20 days after the sol
  • Fig. 5 shows the absorption spectrum of a sol containing a group of synthetic particles and gold particles
  • Raman spectrum shows time course of Raman signal intensity of sol containing pyridin and fine gold particles
  • FIG. 7 shows a calibration curve of an aqueous solution containing a dispersed complex and pyridine.
  • Fine gold particles were synthesized in the aqueous solution by a chemical reduction method in which sodium citrate was added to 120 cc of a 0.6 mM aqueous chloroauric acid solution to a concentration of 1.6 mM.
  • the average particle size of the gold particles was measured by the small-angle X-ray scattering method. It was 0 nm.
  • the solution was divided into four containers, and sodium chloride was added as a coagulant to each container at 50 mM / L to start aggregation.
  • the color tone of the gold fine particle liquid changed from the initial red to reddish purple, bluish purple, reddish brown, brown, black, and finally precipitated.
  • a slurry of synthetic smectite (Laporte) was added to each of the second to fourth containers after a predetermined time. Then, the change of color tone stopped, and a dispersed complex showing a different color tone depending on the ignition timing of the synthetic smectite was obtained.
  • the elapsed time from the addition of sodium chloride to the addition of synthetic smectite is as follows: 2nd container (hereinafter agglomerated state A) ⁇ 3rd container (hereinafter agglomerated state B) ⁇ 4th container (hereinafter agglomerated state C) ).
  • the absorbance of the synthetic smectite alone was measured.
  • the color tone of the gold colloid depends on the state of aggregation of the particles (NG Kh Stammsov, VA Bogatyrev, LA Dykman, and AG Melnikov, J. Colloid Interface Sci., 180 (2), 436- 445 (1996); Spectral Extinction of Colloidal Gold and Its Biospecific Conjugates.), And the results shown in FIG. 1 indicate that the method of the present invention can produce a dispersed complex in which the population state of gold fine particles is controlled. . Evaluation of stability of monodisperse composite sol 1
  • a dispersed complex was prepared in the same manner as in the aggregation state A in the above Preparation Example, except that montmorillonite (manufactured by Kunimine Industry Co., Ltd.) was used instead of the synthetic smectite. Even when left at room temperature, the color tone did not change for several minutes, but gradually turned brown over several days. However, no precipitation occurred.
  • Figure 3 shows the absorbance spectra immediately after preparation, 1 minute, and 20 days later. As shown in Fig. 3, it was found that even in the dispersed complex containing montmorillonite, the population of fine gold particles was stabilized while maintaining a controlled state.
  • the dispersion complex (aggregation state B) of the above sol preparation example was stored at room temperature, and after a predetermined number of days, the pyridine Raman spectrum was measured by the method of Example 1, and the transition of the signal intensity of ring respiratory oscillation was measured. Then, the plot was plotted against the storage period of the dispersed complex (Okinaji in the figure), and Fig. 6 was obtained. As a control, the fourth mixed solution of Example 2 was used (marked by X in the figure). As shown in FIG. 6, it was found that the above-mentioned dispersed complex served as a substrate for the surface enhancement effect for a long period of time, which was nearly two months.
  • the substance analysis method of the present invention is useful for analyzing trace substances or low-concentration substances.

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Abstract

A method for analyzing a substance, characterized in that prepared is a composite disperse system comprising a dispersed phase of lyophobic fine particles which are present in groups and exhibit the surface enhancement effect and, coexisting therein in a concentration sufficient to cover the surroundings of the lyophobic fine particles, another type of fine particles having a higher dispersibility than that of the lyophobic fine particles, a fluid containing a substance to be determined is contacted with the composite disperse system, and the substance to be determined is analyzed by using an optical instrumentation means. The method can be effectively used for analyzing a trace amount of substance or a substance present in a low concentration.

Description

明 細 書  Specification
物質の分析方法 技術分野 Material analysis method Technical field
本発明は、 振動分光法において表面増強効果を利用するすべての分析 方法に関する。 特に、 タンパク質の水溶液中での機能解析など生体成分 の分析を必要とするライフサイエンス分野、 また、 環境中の有害物質な どの分析を必要とする地球 · 環境分野に関する。 背景技術  The present invention relates to all analytical methods utilizing the surface enhancement effect in vibrational spectroscopy. In particular, it relates to the life science field that requires analysis of biological components such as functional analysis of protein in aqueous solution, and the global and environmental fields that require analysis of harmful substances in the environment. Background art
タンパク質 · 核酸などバイォ機能性物質の生理機能の解明は重要な課 題であるが、 それにも増して、 その物理化学的性質の制御と設計が、 バ ィォリアクター、 バイオセンサ一、 D N Aチップ、 近未来的にはバイオ 素子などの実現に不可欠である。 そのため、 バイオ機能性物質の物理化 学的性質の測定方法や、 測定のための分析用試薬の発展が期待されてい る。  Elucidation of the physiological functions of bio-functional substances such as proteins and nucleic acids is an important issue, but control and design of their physicochemical properties are even more important than bioreactors, biosensors, DNA chips, and the near future. Essentially, it is indispensable for realizing bio-elements. Therefore, development of methods for measuring the physicochemical properties of biofunctional substances and analytical reagents for measurement are expected.
バイオ機能性物質は生体中で活性を持ち、 その生体は水を媒体にして いる。 したがって、 その物理化学的性質の解明もまた水溶液中で実施さ れる必要がある。 水中においてもバイォ機能性物質の構造や基質分子と の相互作用を調べる有用な分析方法に、 振動分光法の一種であるラマン 分光がある。  Biofunctional substances have activity in living organisms, which use water as a medium. Therefore, elucidation of its physicochemical properties also needs to be performed in aqueous solution. Raman spectroscopy, a type of vibrational spectroscopy, is a useful analytical method for examining the structure of bio-functional substances and their interaction with substrate molecules even in water.
しかし、 通常のラマン分光では得られるシグナル強度が著しく低く感 度が悪いので、 数％以上の試料濃度を必要としていた。 そのためバイオ 機能性物質の場合、 試料の濃縮操作は必要不可欠であり、 コス トおよび 操作中の試料の逸失や変性の危険が問題となっていた。  However, since the signal intensity obtained by ordinary Raman spectroscopy is extremely low and the sensitivity is poor, a sample concentration of several percent or more was required. Therefore, in the case of biofunctional substances, the operation of concentrating the sample is indispensable, and the cost and the risk of loss or denaturation of the sample during the operation have been a problem.
一方、ラマン分光であっても、試料が金属微粒子と相互作用するとき、 シグナル強度が増幅する表面増強効果が知 られてい る （ "Surface Enhanced Raman Scattering", ed. by L K. Chang and T. E. Fur tak, (Plenum Publishing, N. Y. , 1982))。 その増強感度は通常 1万倍から 1 0 0万倍であると言われている。 On the other hand, even in Raman spectroscopy, when a sample interacts with metal fine particles, It is known that the signal enhances the surface intensifying effect ("Surface Enhanced Raman Scattering", ed. By LK Chang and TE Furtak, (Plenum Publishing, NY, 1982)). It is said that the enhancement sensitivity is usually 10,000 to 100,000 times.
表面増強効果は、 上記の金属微粒子が凝集状態で用いられるとき大き く現れる。 表面増強効果を利用した測定法は、 バイオテクノロジーの研 究手段としても重要である （K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, and M. S. Fe Id, Biomedical Applications of Lasers, 77 (7), 915-924 (1999) ； Surface-enhanced Raman scattering ： A new tool for biomedical spec t roscopy)。  The surface enhancing effect is remarkably exhibited when the metal fine particles are used in an aggregated state. Measurement methods using the surface enhancement effect are also important as biotechnology research tools (K. Kneipp, H. Kneipp, I. Itzkan, RR Dasari, and MS Fe Id, Biomedical Applications of Lasers, 77 (7 ), 915-924 (1999); Surface-enhanced Raman scattering: A new tool for biomedical spectroscopy).
貴金属微粒子の凝集を用いた最近の実験では、 一分子検出を可能にす る 1 0 0 兆倍にも達する表面増強効果が確認された （K. Kneipp, H. Kneipp, R. Manoharan, E. Hani on, I. Itzkan, R. R. Dasari, and M. S. Feld, Applied Spectroscopy, 52 (12) , 1493-1497 (1998) ； Extremely large enhancement factors in surface-enhanced Raman scattering for molecules on colloidal gold clusters)。 このような金属微粒子は表面 増強効果の基質と呼ばれる。 ガリウムやガリウム砒素などの半導体も同 様に基質となり うる。  Recent experiments using agglomeration of precious metal microparticles have confirmed a surface enhancement effect of 100 trillion times that enables single molecule detection (K. Kneipp, H. Kneipp, R. Manoharan, E. Hani on, I. Itzkan, RR Dasari, and MS Feld, Applied Spectroscopy, 52 (12), 1493-1497 (1998); Extremely large enhancement factors in surface-enhanced Raman scattering for molecules on colloidal gold clusters). Such metal fine particles are called substrates for the surface enhancement effect. Semiconductors such as gallium and gallium arsenide can be substrates as well.
表面増強効果の基質として金属微粒子を用いるときの態様としては、 金属微粒子のコロイ ド、金属微粒子を表面に島状に沈積させたフィルム、 ゾルゲル法で金属微粒子を内部に分散させたガラスマトリ ックス、 金属 微粒子を内部に分散させたポリマーマ トリ ックスなどが今まで報告され ている。 又、 本発明者らも特開平 1 1 一 6 1 2 0 9号公報において、 膨 潤性層状ケィ酸塩などの板状微粒子を分散させた分散液中で、' 貴金属微 粒子を還元反応で生成させることによって、 安定な貴金属微粒子の分散 体を得る技術を開示した。 これらの基質のうち、 水溶液中でのナノ貴金属微粒子のコロイ ドが実 用上もっとも便利であるとされる。 その理由として、 1 ) 微粒子が液相 法で合成でき、 取扱いが簡便である。 2 ) 連続流れ分析系への適用がで きる。 3 ) 粒子サイズと形状の制御が可能である。 4 ) 簡単に表面積を 定義できる。 5 ) 理論的解析のために形態を変えられる。 等の利点が指 摘されている （M. Kerker, D. S. Wang, H. Chew. 0. Si iman, and L. A. Bumm, "Surface Enhanced Raman Scattering", ed. by R. K. Chang and T. E. Furtak, (Plenum Publishing, N. Y. , 1982) , pp. 109-128 ； Enhanced Raman scattering by molecules adsorbed at the surface of colloidal particles)。 Examples of the mode of using metal fine particles as a substrate for the surface enhancement effect include a colloid of metal fine particles, a film in which metal fine particles are deposited in an island shape on the surface, a glass matrix in which metal fine particles are dispersed inside by a sol-gel method, A polymer matrix in which metal fine particles are dispersed has been reported so far. Further, the present inventors also disclose in Japanese Patent Application Laid-Open No. Hei 11-116209 that a noble metal fine particle is reduced by a reduction reaction in a dispersion liquid in which plate-like fine particles such as a swellable layered silicate are dispersed. A technique for obtaining a stable dispersion of noble metal fine particles by producing the same has been disclosed. Of these substrates, colloids of nano-noble metal particles in aqueous solution are said to be the most practical in practice. The reasons are: 1) Fine particles can be synthesized by a liquid phase method, and handling is easy. 2) Applicable to continuous flow analysis system. 3) Particle size and shape can be controlled. 4) The surface area can be easily defined. 5) Can change form for theoretical analysis. (M. Kerker, DS Wang, H. Chew. 0. Si iman, and LA Bumm, "Surface Enhanced Raman Scattering", ed. By RK Chang and TE Furtak, (Plenum Publishing, NY, 1982), pp. 109-128; Enhanced Raman scattering by molecules adsorbed at the surface of colloidal particles).
いずれにしても金属微粒子を表面増強効果の基質として用いるために は、 分散状態を安定に保つ必要がある。 従来、 分散状態を制御する方法 としては、 金属微粒子が疎液性微粒子であることから、 1 ) 液相中で安 定剤の添加、 2 ) 固相上へのデポジッ ト （コ一ティ ングを含む）、 3 ) 上 記のようなガラス、 ポリマーなどのマトリ ックス中への包括、 4 )上記特 開平 1 1 一 6 1 2 0 9号公報のように膨潤性層状ケィ酸塩を金属微粒子 と共存させるなどが提案されてきた。  In any case, in order to use metal fine particles as a substrate for the surface enhancement effect, it is necessary to keep the dispersed state stable. Conventionally, the dispersion state has been controlled by the following methods: 1) adding a stabilizer in the liquid phase, and 2) depositing (coating) on the solid phase because the metal fine particles are lyophobic fine particles. 3) Incorporation of glass, polymer, etc. into the matrix as described above, 4) Swellable layered silicate as fine metal particles as disclosed in JP-A-11-161209. It has been proposed to coexist.
これらの制御法のうち液相中で使われる安定剤としては、 ドデシル硫 酸ナトリウムなどの界面活性剤、 ポリ ビニルアルコール、 ポリ ビニルビ リジン、 ポリエチレングリコール、 N—ビニルピロリ ドン、 ゥシ血清ァ ルブミン、 ァーグロブリ ン、 ゼラチンのような保護コロイ ドが知られて いる。 また、 特開平 0 9 — 0 7 0 5 2 7 「コロイ ド凝集の防止方法」 に は卜リスヒ ドロキシメチルァミノメタンなどの緩衝剤の安定化作用が開 示されている。  Among these control methods, the stabilizers used in the liquid phase include surfactants such as sodium dodecyl sulfate, polyvinyl alcohol, polyvinyl pyridine, polyethylene glycol, N-vinyl pyrrolidone, sera albumin, and var. Protective colloids such as gelatin and gelatin are known. Further, Japanese Patent Application Laid-Open No. 09-070527 "Method for Preventing Colloid Aggregation" discloses a stabilizing effect of a buffer such as trishydroxymethylaminoaminomethane.
また、 固相上へのデポジッ トとしては、 ガラス板へ微粒子をデポジッ トして凝集をある程度の段階で止める方法がよく用いられる。 これによ れば液相法で合成したナノ微粒子をガラス上に沈積させて、 サイズや形 態の異なる凝集を生成させることが可能である。 As a method of depositing on a solid phase, a method of depositing fine particles on a glass plate and stopping aggregation at a certain stage is often used. This In this case, nanoparticles synthesized by the liquid phase method can be deposited on glass to form aggregates of different sizes and shapes.
しかし、 水溶液中でのナノ貴金属微粒子のコロイ ドのように、 液体を 分散媒とする疎液性微粒子の分散相中で、 微粒子の機能性を保ったまま 凝集状態を沈殿させずに液中にとどめておくのは安定化剤を用いたとし てもきわめて困難であった。 その結果、 疎液性微粒子の凝集状態に依存 する表面増強効果の基質の製法の再現性や安定性は乏しく、 性能もまだ 不十分であった。  However, like a colloid of nano-noble metal particles in an aqueous solution, in a dispersed phase of lyophobic particles using a liquid as a dispersion medium, the particles remain in the liquid without precipitating the aggregated state while maintaining the functionality of the particles. It was extremely difficult to keep, even with stabilizers. As a result, the reproducibility and stability of the substrate production method of the surface enhancement effect depending on the aggregation state of the lyophobic fine particles were poor, and the performance was still insufficient.
また、 従来の安定化剤は、 微粒子の表面に付着し微粒子どうしの接近 を抑制させて凝集を防止するため、 金属微粒子の重要な機能である表面 活性が失われた。 金属微粒子を固相上にデポジッ トしても、 凝集サイズ 分布の分散や偏差が大きく製法の再現性に乏しく、 しかも不安定であつ てチオール等の有機単分子層でコーティ ングしても数日程度の安定性に 止まっていた。 マトリ ックス中への包括では、 マ 卜リ ックスによる金属 微粒子の表面活性の喪失や、 マトリ ックス中の物質移動速度の低下が生 じ、 優れた表面増強効果の基質は得られなかった。 又、 特開平 1 1 一 6 1 2 0 9号公報に記載の技術は、 板状微粒子を分散させた分散液中で金 属微粒子を生成するために金属微粒子の生成に長時間を要しコス ト高と なる上、 分解しやすいァセトジカルボン酸を還元剤に用いるので取扱が 不便である。  In addition, conventional stabilizers adhere to the surface of the fine particles and suppress the approach of the fine particles to prevent agglomeration, so that the surface activity, which is an important function of the fine metal particles, is lost. Even when metal fine particles are deposited on a solid phase, the dispersion and deviation of the aggregate size distribution are large, the reproducibility of the manufacturing method is poor, and it is unstable, and it is unstable for several days even if coated with an organic monolayer such as thiol. The degree of stability was limited. Incorporation into the matrix resulted in the loss of the surface activity of the metal microparticles due to the matrix and the decrease in the mass transfer rate in the matrix, and a substrate having an excellent surface enhancement effect could not be obtained. In addition, the technology described in Japanese Patent Application Laid-Open No. H11-20909 requires a long time to generate metal fine particles in order to generate metal fine particles in a dispersion liquid in which plate-like fine particles are dispersed, and thus requires a long time. In addition, it is inconvenient to handle because the decomposed acetodicarboxylic acid is used as the reducing agent.
即ち、 疎液性微粒子の表面活性を高く維持しながら、 再現性よく製造 でき、 長期間安定で、 迅速な応答を与える表面増強効果の基質は今まで 知られていない。  That is, a substrate having a surface enhancing effect that can be produced with good reproducibility while maintaining the surface activity of the lyophobic fine particles at a high level, and is stable for a long time and gives a quick response has not been known.
それゆえ、 この発明の目的は、 凝集のように群をなして存在する疎液 性微粒子を分散相として止め、 得られた分散複合体を実用的な表面増強 効果の基質として用いる分析方法を提供することにある。 発明の開示 Therefore, an object of the present invention is to provide an analysis method in which lyophobic fine particles present in a group such as agglomeration are stopped as a dispersed phase, and the obtained dispersed complex is used as a substrate for a practical surface enhancement effect. Is to do. Disclosure of the invention
上記目的を達成するために、 この分析方法は、 群をなして存在する疎 液性微粒子の分散相中に、疎液性微粒子よりも分散性の高い微粒子（以下 「分散性微粒子」 という。 ）を、 疎液性微粒子群の周囲を覆うに十分にた る濃度で共存させて得られた分散複合体を、 表面増強効果の基質として 用いる。そして、分散複合体と、測定対象物質を含む流体とを接触させ、 測定対象物質が疎液性微粒子群に接近して得られる表面増強効果を利用 し、 測定対象物質の濃度あるいは性質を光学的計測手段で測定すること を特徴とする。  In order to achieve the above object, this analysis method uses fine particles having higher dispersibility than lyophobic fine particles (hereinafter referred to as “dispersible fine particles”) in a dispersed phase of lyophobic fine particles present in groups. And a dispersion complex obtained by coexisting at a concentration sufficient to cover the periphery of the lyophobic fine particle group is used as a substrate for the surface enhancement effect. Then, the dispersion complex is brought into contact with the fluid containing the substance to be measured, and the concentration or property of the substance to be measured is optically measured by utilizing the surface enhancement effect obtained when the substance to be measured approaches the lyophobic fine particle group. It is characterized by measurement by measuring means.
本発明によれば、 疎液性微粒子の集団状態が分散性微粒子によって維 持されるので、 凝集が必要以上に進行したり沈殿を生じたりすることを 抑制できる。 その結果、 表面増強効果の基質としての性能が長期間維持 され、 表面増強効果を用いた分析を簡便に実施できる。 発明の詳細な開示  According to the present invention, the collective state of the lyophobic fine particles is maintained by the dispersible fine particles, so that it is possible to suppress aggregation from advancing more than necessary or causing precipitation. As a result, the performance of the surface enhancement effect as a substrate is maintained for a long period of time, and analysis using the surface enhancement effect can be easily performed. Detailed Disclosure of the Invention
本発明において分散複合体は通常、 下記 （ a ) ~ ( d ) の工程を含む 方法で製造される。  In the present invention, the dispersion complex is usually produced by a method including the following steps (a) to (d).
( a ) 疎液性微粒子を液相に分散させる工程。  (a) a step of dispersing lyophobic fine particles in a liquid phase;
( b ) 凝集剤を添加するなどして凝集を開始させ、 疎液性微粒子の群 を得る工程。  (b) A step of initiating aggregation by adding an aggregating agent or the like to obtain a group of lyophobic fine particles.
( c ) (b)の工程の後に、 分散性微粒子を、 疎液性微粒子群の周囲を覆 うに十分にたる濃度となるように加える工程。  (c) a step of adding the dispersible fine particles after the step (b) to a concentration sufficient to cover the periphery of the lyophobic fine particles.
( d ) 得られた疎液性微粒子の群を分散複合体として回収する工程。 疎液性微粒子としては、 特に限定されないが、 粒径が原子サイズに近 い 1 〜 1 0 0 n mの金、 銀、 銅、 白金、 ニッケル、 インジウム、 パラジ ゥムから選ばれる少なく ともひとつ以上の金属を主成分とする金属微粒 子やガリウム、 ガリウム砒素などの半導体微粒子を用いてよい。 しかし これに限定されるものではなく、 バルクとは異なる機能が発現する微粒 子であれば用いてよい。 (d) a step of collecting the obtained group of lyophobic fine particles as a dispersed complex. The lyophobic fine particles are not particularly limited, but gold, silver, copper, platinum, nickel, indium, palladium having a particle size close to the atomic size of 1 to 100 nm. Metal fine particles containing at least one metal selected from the group consisting of at least one metal and semiconductor fine particles such as gallium and gallium arsenide may be used. However, the present invention is not limited to this, and any fine particles that exhibit a function different from that of bulk may be used.
これらの疎液性微粒子は、 特に限定はされないが、 液相法で合成しそ のまま上記（ b )工程に供することができる。 また、 別の方法で得た微粒 子を液中に加え撹拌させるなどして、 液相に分散させてもよい。  These lyophobic fine particles are not particularly limited, but can be synthesized by a liquid phase method and subjected to the above step (b) as it is. Further, fine particles obtained by another method may be added to a liquid and stirred to be dispersed in a liquid phase.
凝集を開始させ、疎液性微粒子群を得る方法は、特に限定されないが、 粒子濃度を上げる、 塩化ナトリ ウムや硫酸アルミニウムなどのように塩 析現象をもたらす電解質を加えイオン強度を上げる、 ポリマーで架橋さ せる、 温度を高くする、 分散媒の極性を低くするなどの手段を選ぶこと ができる。  The method of initiating aggregation and obtaining a lyophobic fine particle group is not particularly limited, but includes increasing the particle concentration, increasing the ionic strength by adding an electrolyte that causes a salting-out phenomenon such as sodium chloride or aluminum sulfate, or using a polymer. Means such as crosslinking, increasing the temperature, and decreasing the polarity of the dispersion medium can be selected.
液中の微粒子分散相の安定性は D L V O理論で説明される。 金微粒子 を例に D L V O理論による説明を以下に記述するが、 他の疎液性微粒子 でも同様に説明される。 化学的還元を受けた金微粒子には、 還元剤ァニ オンゃ錯体金属ァニオンが吸着され負電荷を帯びる （M. A. Haya t Ed. " Co I l o i da l Go l d " vo l . 1 and vo l . 2, Ac ademi c Pre s s Inc. , 1984)。 この 静電的な反発ポテンシャルと、 ファンデルワールス力等に依存する引力 ポテンシャルとの相対的な大きさのバランスが適当であると、 総ポテン シャル曲線には極大が現れる。 微粒子の運動エネルギーがその極大より も大きくなければ、 微粒子はその極大を越えて互いに接近することがで きず凝集が起こらないので系は安定化する。 一方、 対イオンの吸着等や イオン強度の増加によって静電的な反撥ポテンシャルが変化すると、 総 ポテンシャル曲線の極大値が減少し、 粒子はエネルギー障壁を越えて凝 集するようになる。 一方では複数の微粒子を架橋しうるポリマーの存在 によって架橋凝集が発生する。 疎液性微粒子よりも分散性の高い微粒子とは、 凝集を開始させた状況 において、 前述の D L V O理論における総ポテンシャル曲線に現れる極 大の位置が、 機能性微粒子の極大の位置が示す粒子間距離よりも、 広く 離れた粒子間距離を示す微粒子である。 あるいは、 疎液性微粒子より も 分散性の高い微粒子とは、 疎液性微粒子の総ポテンシャル曲線の極大が 消滅しつつある状況において、 十分な粒子間距離を示す位置に、 十分大 \きな極大が現れる総ポテンシャル曲線を持った微粒子である。 総ポテン シャル曲線の形は、 主として Hamake r定数と S t e rn電位の値によって決 定されるので、 この定義において特に疎液性微粒子の種類が限定される ものではない。 The stability of the dispersed phase of fine particles in a liquid is explained by DLVO theory. The explanation based on the DLVO theory will be described below using gold particles as an example, but the same applies to other lyophobic particles. The gold particles subjected to the chemical reduction are adsorbed with a reducing agent anion-complex metal anion and become negatively charged (MA Hayat Ed. "Co Iloi da l Gold" vol. 1 and vol. 2, Acemic Press Inc., 1984). If the relative magnitude of the electrostatic repulsion potential and the attractive potential depending on the van der Waals force, etc. is appropriate, the maximum appears in the total potential curve. If the kinetic energy of the particles is not greater than its maximum, the system will stabilize because the particles will not be able to approach each other beyond the maximum and will not aggregate. On the other hand, when the electrostatic repulsion potential changes due to adsorption of counter ions or an increase in ionic strength, the maximum value of the total potential curve decreases, and the particles aggregate over the energy barrier. On the other hand, cross-linking aggregation occurs due to the presence of a polymer capable of cross-linking a plurality of fine particles. Fine particles with higher dispersibility than lyophobic fine particles mean that the position of the maximum that appears in the total potential curve in the above-mentioned DLVO theory is the distance between particles that is indicated by the position of the maximum of the functional fine particles when the aggregation starts. It is a fine particle that shows the distance between particles far apart from each other. Alternatively, the fine particles having a higher dispersibility than the lyophobic fine particles refer to a sufficiently large local maximum at a position showing a sufficient interparticle distance in a situation where the maximum of the total potential curve of the lyophobic fine particles is disappearing. Are fine particles having a total potential curve. Since the shape of the total potential curve is mainly determined by the values of the Hamaker constant and the Stem potential, this definition does not particularly limit the type of lyophobic fine particles.
このような分散性微粒子の具体的な例としては、 スメクタイ トなどの 膨潤性層状ケィ酸塩を挙げることができる。  Specific examples of such dispersible fine particles include swellable layered silicates such as smectite.
膨潤性層状ケィ酸塩が分散性微粒子の例として特に挙げられる理由は そのフ： Γ—ス面が負電荷を帯びており、 疎液性微粒子が凝集して沈積す るような状況においても、 十分分散し続けるからである。 さらに過酷な 状況で沈積しても、 ある程度までは緩やかな凝集形態を保ち、 撹拌等の 剪断力で容易に再分散するからである。 また粒径が小さく、 わずかな重 量濃度でも、 充分な個数が分散液中に存在し、 疎液性微粒子の周辺を覆 う ことができるからである。 また形状が平板上であるため、 粒子間相互 作用が生じやすく、 分散媒の見かけの粘度を上げ、 粒子の拡散速度を下 げ、 疎液性微粒子の凝集の進行を妨げるからである。  The reason why the swellable layered silicate is particularly cited as an example of the dispersible fine particles is as follows: Even in a situation where the base surface is negatively charged and the lyophobic fine particles aggregate and deposit. This is because they are sufficiently dispersed. Even if sedimentation is more severe, it maintains a loose coagulation form to some extent and is easily redispersed by shearing force such as stirring. In addition, even if the particle diameter is small and a slight weight concentration is obtained, a sufficient number is present in the dispersion liquid and can cover the periphery of the lyophobic fine particles. Also, because the shape is on a flat plate, interaction between particles is likely to occur, increasing the apparent viscosity of the dispersion medium, decreasing the diffusion speed of the particles, and hindering the progress of aggregation of the lyophobic fine particles.
層状層状ケィ酸塩は、 合成物、 天然物に限らず使用できるが、 好まし くは合成物が用いられる。 天然物を精製して得られる膨潤性粘土鉱物も 好ましく用いられる。 合成物は、 天然物とは異なり、 化学的に均一で不 純物が少なく、 凝集性のイオンを含まず膨潤性が高く、 さらに層間に鉄 等の有色の金属を含まず透明度が高いため、 光学的計測手段に向くから である。 The layered layered silicate can be used without being limited to a synthetic or natural product, but a synthetic product is preferably used. Swellable clay minerals obtained by purifying natural products are also preferably used. Unlike natural products, synthetic products are chemically uniform, contain few impurities, contain no cohesive ions, have high swelling properties, and do not contain colored metals such as iron between layers and have high transparency. Suitable for optical measuring means It is.
このような膨潤性層状ケィ酸塩は市販されている。  Such swellable layered silicates are commercially available.
本発明における分散相の分散媒は、 水、 アルコール、 炭化水素などの 液体あるいは気体など、 特に限定されないが、 水を好ましく用いること ができる。 このとき、 この発明の分散複合体は、 水を分散媒とするゾル として回収される。 また、 この発明の分散複合体は、 乾燥などの手段に よりゲルとして回収される。  The dispersion medium of the disperse phase in the present invention is not particularly limited, such as liquid, gas such as water, alcohol, and hydrocarbon, but water can be preferably used. At this time, the dispersion composite of the present invention is recovered as a sol using water as a dispersion medium. Further, the dispersed complex of the present invention is recovered as a gel by means such as drying.
本発明における分散性微粒子の濃度は、 制御すべき疎液性微粒子の周 囲を覆うに十分にたる濃度と見なされれば、 特に限定されない。 こ こで 周囲を覆うという概念は、 単位空間内に存在する疎液性微粒子の個数よ りも、 分散性の高い微粒子の個数が大過剰にあることを意味し、 結果と して制御すべき疎液性微粒子の凝集の進行や沈殿を抑制するにたるもの であればよい。 例えば、 疎液性微粒子の個数濃度が 1 0 0 0 c c 中に約 1 0の 1 2乗個であるとき、 分散性微粒子は 1 0の 1 5乗個以上あれば 十分であると考えてよい。 具体的な例をあげると、 分析の実施に必要な 疎液性微粒子の好ましい濃度は 0 . 0 1 m M〜 4 M、この範囲濃度の疎液 性微粒子を覆うに必要な分散性微粒子の濃度は膨潤性層状ケィ酸塩の場 合で通常 0 . 1 g / L以上である。 一方、 膨潤性層状ケィ酸塩からなる分 散性微粒子の調整可能な最高濃度は通常 2 5 O g Z Lで、 濃縮した場合 3 0 0 g / Lである。 しかしこれに限定されるわけではなく、 疎液性微 粒子の種類と濃度、 凝集を開始させる手段、 そして分散性微粒子自体の 性質に依存する。  The concentration of the dispersible fine particles in the present invention is not particularly limited as long as the concentration is considered to be sufficient to cover the periphery of the lyophobic fine particles to be controlled. The concept of covering the periphery here means that the number of fine particles with high dispersibility is much larger than the number of lyophobic fine particles existing in the unit space, and should be controlled as a result. Any material can be used as long as it suppresses the progress of aggregation and precipitation of the lyophobic fine particles. For example, when the number concentration of lyophobic fine particles is about 10 12 in 100 cc, it may be sufficient if the number of dispersible fine particles is 10 15 or more. . As a specific example, the preferred concentration of the lyophobic fine particles required for performing the analysis is 0.01 mM to 4 M, the concentration of the dispersible fine particles required to cover the lyophobic fine particles in this range. Is usually 0.1 g / L or more in the case of a swellable layered silicate. On the other hand, the maximum adjustable concentration of the dispersible fine particles composed of the swellable layered silicate is usually 25 Og ZL, and 300 g / L when concentrated. However, it is not limited to this, but depends on the type and concentration of the lyophobic fine particles, the means of initiating aggregation, and the properties of the dispersible fine particles themselves.
本発明によつて疎液性微粒子の分散状態を制御することができる。 本 発明のように集団状態の疎液性微粒子を制御し、 安定化した例は今まで 知られていない。 また本発明のように、 一ヶ月以上安定化した例も知ら れていない。 本発明によって分散状態が制御された疎液性微粒子の分散 複合体が得られる。 この分散複合体では、 機能発現に好ましい状態で疎 液性微粒子の集団が存在しており、 光学素子、 センサ一、 触媒などの利 用が考えられる。 According to the present invention, the dispersion state of the lyophobic fine particles can be controlled. An example of controlling and stabilizing the lyophobic fine particles in a collective state as in the present invention has not been known so far. Further, there is no known example of stabilization for one month or more as in the present invention. Dispersion of lyophobic fine particles whose dispersion state is controlled by the present invention A complex is obtained. In this dispersed composite, a group of lyophobic fine particles exists in a state that is favorable for the expression of functions, and the use of optical elements, sensors, catalysts, and the like is conceivable.
本発明における測定対象物質としては、 水溶液中のアミノ酸、 塩基、 タンパク質、 核酸が挙げられる。 また環境中の、 芳香族塩素化合物など が挙げられる。 しかしこれに限定されるものではない。  Examples of the substance to be measured in the present invention include amino acids, bases, proteins, and nucleic acids in an aqueous solution. Also, there are aromatic chlorine compounds in the environment. However, it is not limited to this.
本発明における光学的計測手段には、 R a m a n分光法、 赤外分光法 などの振動分光法を用いることができる。 表面増強効果を利用した光学 的計測手段は、 RAS (Reflection absorption spectroscopy)、 SEWS (Surface electromagnet ic wave spectroscopy) , SE IRA (Surf ace-enhanced infrared spectroscopy)、 SERS (Surface-enhanced Raman spectroscopy)、 SERRS ( Surface-enhanced resonance Raman spectroscopy) , SEHRS (Surface-enhanced hyper Raman scat ter ing)等力 知られてレ る。  Vibration spectroscopy, such as Ramn spectroscopy and infrared spectroscopy, can be used for the optical measurement means in the present invention. Optical measurement methods using the surface enhancement effect include RAS (Reflection absorption spectroscopy), SEWS (Surface electromagnetic wave spectroscopy), SE IRA (Surf ace-enhanced infrared spectroscopy), SERS (Surface-enhanced Raman spectroscopy), SERRS ( Surface-enhanced resonance Raman spectroscopy) and SEHRS (Surface-enhanced hyper Raman scatting) are well known.
この発明の分析方法において、 疎液性微粒子には、 振動分光に用いら れる波長範囲で表面増強効果を示すことが確認された金、銀、銅、 白金、 ニッケル、インジウム、パラジウム等の金属微粒子、 あるいはガリウム、 ガリウム砒素などの半導体微粒子を好ましく用いることができる。 この 発明の分析方法において、 分散性微粒子には、 透光性の良い合成スメク タイ トを好ましく用いることができる。  In the analysis method of the present invention, the lyophobic fine particles include metal fine particles such as gold, silver, copper, platinum, nickel, indium, and palladium, which have been confirmed to exhibit a surface enhancing effect in a wavelength range used for vibrational spectroscopy. Alternatively, semiconductor fine particles such as gallium and gallium arsenide can be preferably used. In the analysis method of the present invention, a synthetic smectite having good translucency can be preferably used as the dispersible fine particles.
本発明の分散複合体では流れ系の管壁や容器の壁に金属微粒子は吸着 せず、 少なく とも数ケ月間の間、 表面増強効果の基質として働くので、 流れ系での分析を実施することができる。 分散複合体がゾルのとき、 表 面増強効果の基質として流れ系の中に流し、 測定対象物質を含む試料溶 液と接触させ、 光学的計測手段で測定することができる。 このような流 れ系の例にはキヤピラリー電気泳動、 各種のクロマトグラフィーが知ら れている。 また、 分散複合体がゲルのとき、 センサ一のように測定対象物質を含 む試料溶液と接触させて用いることができる。 好ましくは、 この分散複 合体を使い捨てのセンサ一として用いることができる。 もちろんこのセ ンサ一は流れ系の一部を構成していてもよい。 In the dispersion composite of the present invention, metal particles are not adsorbed on the pipe wall or vessel wall of the flow system, and serve as a substrate for the surface enhancement effect for at least several months. Can be. When the dispersed complex is a sol, it can be flowed into a flow system as a substrate for the surface enhancement effect, brought into contact with a sample solution containing the substance to be measured, and measured by optical measurement means. Examples of such a flow system include capillary electrophoresis and various types of chromatography. Further, when the dispersion complex is a gel, it can be used by being brought into contact with a sample solution containing the substance to be measured like a sensor. Preferably, this dispersion composite can be used as a disposable sensor. Of course, this sensor may form part of a flow system.
本発明の分散複合体においては、 合成スメクタイ トに配位子化合物、 抗体、 抗原、 酵素、 酵素基質、 核酸、 核酸の補体からなる群から選ばれ るひとつ以上の物質を修飾し、 測定対象物質を認識ないしは配向させる 機能を持たせることができる。 この場合、 表面増強効果は疎液性微粒子 の表面からの距離が離れると増感度が著しく低下するため、 認識された 物質あるい,は配向による一部の官能基のみが、 表面増強効果に与ること ができ、 選択的な物質の測定方法に利用できる。 図面の簡単な説明  In the dispersion complex of the present invention, the synthetic smectite is modified with one or more substances selected from the group consisting of a ligand compound, an antibody, an antigen, an enzyme, an enzyme substrate, a nucleic acid, and a nucleic acid complement, and It can have the function of recognizing or orienting substances. In this case, the surface enhancement effect is significantly reduced when the distance from the surface of the lyophobic fine particles is increased, so that only the recognized substance or some of the functional groups due to the orientation have an effect on the surface enhancement effect. It can be used for selective substance measurement. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 金微粒子群及び合成スメクタイ トを含むゾルの吸光スぺク ト ル ； 図 2は、 同ゾルの 2 0 日後の吸光スぺク トル ； 図 3は、 金微粒子群 及びモンモリ 口ナイ トを含むゾルの吸光スぺク トル ； 図 4は、 合成スメ ク夕ィ ト及び金微粒子群を含む乾燥ゲルの吸光スぺク トル ； 図 5は、 ピ リジン及び金微粒子群を含むゾルのラマンスぺク トル ； 図 6は、 ピリジ ン及び金微粒子群を含むゾルのラマンシグナル強度の経時推移；図 7は、 分散複合体とピリジンを含む水溶液の検量線である。 発明を実施するための最良の形態  Fig. 1 shows the absorption spectrum of the sol containing the fine gold particles and synthetic smectite; Fig. 2 shows the light absorption spectrum of the sol 20 days after the sol; Absorption spectrum of a sol containing synthetic particles and a group of fine particles of gold; Fig. 5 shows the absorption spectrum of a sol containing a group of synthetic particles and gold particles; Raman spectrum; FIG. 6 shows time course of Raman signal intensity of sol containing pyridin and fine gold particles; FIG. 7 shows a calibration curve of an aqueous solution containing a dispersed complex and pyridine. BEST MODE FOR CARRYING OUT THE INVENTION
-分散複合体ゾルの調製例一  -Preparation example of dispersion complex sol 1
0 . 6 m Mの塩化金酸水溶液 1 2 0 c c にクェン酸ナトリウムを 1 . 6 m Mとなるように加える化学還元法によって、 金微粒子を水溶液中で 合成した。 金微粒子の平均粒径を小角 X線散乱法で測定した結果、 約 4 0 n mであった。 得られた金微粒子含有水溶液について吸光度を測定し た後、 4つの容器に分け、 各容器に凝集剤として塩化ナトリウムを 5 0 mM/ Lとなるように入れて凝集を開始させた。 Fine gold particles were synthesized in the aqueous solution by a chemical reduction method in which sodium citrate was added to 120 cc of a 0.6 mM aqueous chloroauric acid solution to a concentration of 1.6 mM. The average particle size of the gold particles was measured by the small-angle X-ray scattering method. It was 0 nm. After measuring the absorbance of the obtained aqueous solution containing fine gold particles, the solution was divided into four containers, and sodium chloride was added as a coagulant to each container at 50 mM / L to start aggregation.
すると第 1 の容器内では金微粒子の液の色調は、当初の赤から、赤紫、 青紫、 赤褐色、 褐色、 黒と変化し、 最後は沈殿していった。 第 2〜第 4 の容器には各々所定時間経過後に合成スメクタイ ト （Laporte 社製）のス ラリーを添加した。 すると、 色調の変化は停止し、 合成スメクタイ トの 点火時期に応じて異なる色調を示す分散複合体が得られた。 塩化ナトリ ゥム添加時から合成スメクタイ ト添加時までの経過時間は、第 2容器（以 下、 凝集状態 A) <第 3容器（以下、 凝集状態 B) <第 4容器（以下、 凝集 状態 C)の順とした。 図 1 に塩化ナトリウム添加前（=凝集前）、 凝集状態 A、 凝集状態 B及び凝集状態 Cの吸光スペク トルを示す。 尚、 対照とし て合成スメクタイ 卜のみの吸光度を測定した。  Then, in the first container, the color tone of the gold fine particle liquid changed from the initial red to reddish purple, bluish purple, reddish brown, brown, black, and finally precipitated. A slurry of synthetic smectite (Laporte) was added to each of the second to fourth containers after a predetermined time. Then, the change of color tone stopped, and a dispersed complex showing a different color tone depending on the ignition timing of the synthetic smectite was obtained. The elapsed time from the addition of sodium chloride to the addition of synthetic smectite is as follows: 2nd container (hereinafter agglomerated state A) <3rd container (hereinafter agglomerated state B) <4th container (hereinafter agglomerated state C) ). Figure 1 shows the absorbance spectra before aggregation (= before aggregation), aggregation state A, aggregation state B, and aggregation state C. As a control, the absorbance of the synthetic smectite alone was measured.
粒径が同じの場合、 金コロイ ドの色調は粒子の凝集状態に依存するの で（N. G. Khlebtsov, V. A. Bogatyrev, L. A. Dykman, and A. G. Melnikov, J. Colloid Interface Sci. , 180 (2) , 436-445 (1996) ； Spectral Extinction of Colloidal Gold and Its Biospecif ic Conjugates. )、 図 1 に示された結果から、 本発明の方法で金微粒子の集団状態を制御し た分散複合体を製造できることがわかった。 一分散複合体ゾルの安定性評価 1 一  If the particle size is the same, the color tone of the gold colloid depends on the state of aggregation of the particles (NG Khlebtsov, VA Bogatyrev, LA Dykman, and AG Melnikov, J. Colloid Interface Sci., 180 (2), 436- 445 (1996); Spectral Extinction of Colloidal Gold and Its Biospecific Conjugates.), And the results shown in FIG. 1 indicate that the method of the present invention can produce a dispersed complex in which the population state of gold fine particles is controlled. . Evaluation of stability of monodisperse composite sol 1
上記調製例における第 1容器内の溶液の吸光スぺク トル、 及び凝集状 態 A~ Cの分散複合体の 2 0 日経過後の吸光スぺク トルを各々図 2に示 す。 図 2に見られるように、 合成スメクタイ トを含む分散複合体では、 金微粒子の集団が制御された状態を保って長期にわたり安定化されてい ることがわかった。 一分散複合体ゾルの安定性評価 2 — FIG. 2 shows the light absorption spectrum of the solution in the first container and the light absorption spectrum of the dispersed complex of the aggregated states A to C after a lapse of 20 days in the above preparation example. As shown in Fig. 2, it was found that in the dispersed complex containing the synthetic smectite, the population of fine gold particles was stabilized for a long time while maintaining a controlled state. Evaluation of stability of monodispersed composite sol 2 —
合成スメクタイ トの代わりにモンモリ ロナイ ト (クニミネ工業株式会 社製）を用いた以外は、上記調製例の凝集状態 Aと同様に分散複合体を調 製した。 室温で放置しても、 数分間は色調に変化は見られなかったが、 数日かけて徐々に褐色に変わっていった。 但し、 沈殿は生じなかった。 調製直後、 1分後及び 2 0 日経過後の吸光スペク トルを図 3に示す。 図 3に見られるように、 モンモリロナイ トを含む分散複合体でも金微粒子 の集団が制御された状態を保って安定化されていることがわかつた。  A dispersed complex was prepared in the same manner as in the aggregation state A in the above Preparation Example, except that montmorillonite (manufactured by Kunimine Industry Co., Ltd.) was used instead of the synthetic smectite. Even when left at room temperature, the color tone did not change for several minutes, but gradually turned brown over several days. However, no precipitation occurred. Figure 3 shows the absorbance spectra immediately after preparation, 1 minute, and 20 days later. As shown in Fig. 3, it was found that even in the dispersed complex containing montmorillonite, the population of fine gold particles was stabilized while maintaining a controlled state.
—分散複合体ゲルの調製例と安定性評価一 —Preparation example and stability evaluation of dispersed composite gel
紫外線照射により表面を親水化したポリスチレン製プレートを準備し. 上記ゾル調製例で調製した凝集状態 A ~ Cの分散複合体をそのプレー卜 上に滴下し、 乾燥してゲル化させた。 比較のために、 合成スメクタイ ト を含まない金微粒子含有溶液も同様にプレート上に滴下した。 合成スメ クタイ トを含むゲルでは、 金微粒子は赤褐色ないしは褐色の色調を保つ てプレート上に均一に拡がっていた。 この状態は少なく とも 6ヶ月安定 であった。 一方、 合成スメクタイ トを含まない溶液に由来する金微粒子 は黒色沈積物としてプレート上に不均一に拡がっていた。 確認のため、 プレート上に得られた固体の分散複合体の吸光スペク トルを図 4に示す ( 図 4に見られるように、 合成スメクタイ 卜の存在により金微粒子の集団 が制御された状態を保って安定化されていることがわかった。 一実施例 1 一 A plate made of polystyrene whose surface was hydrophilized by ultraviolet irradiation was prepared. The dispersed complex in the aggregated state A to C prepared in the above sol preparation example was dropped on the plate, dried and gelled. For comparison, a solution containing fine gold particles containing no synthetic smectite was similarly dropped on the plate. In the gel containing the synthetic smectite, the fine gold particles were uniformly spread on the plate while maintaining a reddish brown or brown color. This condition was stable for at least six months. On the other hand, the fine gold particles derived from the solution containing no synthetic smectite spread unevenly on the plate as black deposits. For confirmation, the absorption spectrum of the solid dispersion complex obtained on the plate is shown in Fig. 4 (As shown in Fig. 4, the state where the population of gold particles was controlled by the presence of synthetic smectite was maintained. Example 1 Example 1
上記ゾル調製例の分散複合体 （凝集状態 B ) を調製後、 その 5 7 0 μ 1 を採り、 0 · 0 5 Μのピリジン水溶液 3 0 1 とよく混合させ、 第 1 混合液とした。 別途、 純水 5 7 0 a 1 を採り、 0. 0 5 Mのピリジン水 溶液 3 0 i 1 とよく混合させ、 第 2混合液とした。 これら 2種の混合液 について、 R a m a nモジュールを備えたフーリエ変換赤外分光装置 N i c o 1 e t M a g n a 6 5 0を用いて、 励起波長 1 0 6 4 nmで ラマンスペク トルを測定した。 After preparing the dispersion complex (aggregation state B) of the above sol preparation example, take 570 μl thereof, mix it well with 0. A mixed solution was obtained. Separately, pure water 570 a1 was taken and mixed well with a 0.05 M aqueous pyridine solution 30 i 1 to obtain a second mixed solution. The Raman spectrum of these two mixed solutions was measured at an excitation wavelength of 1064 nm using a Fourier transform infrared spectrometer Nico 1 et Magna650 equipped with a Raman module.
その結果、 第 1混合液では、 2. 5 m Mのピリジンの環呼吸振動 （約 1 0 1 0 c m—1) が強く表れた。 一方、 第 2混合液（通常の R am a n 分光）ではこの濃度のピリジンをまったく観察できなかった。 よって、 上 記分散複合体は、 市販のラマン分光器を用いて簡便に表面増強効果を用 いた分析を可能とする、 表面増強効果の基質となることがわかった。 尚、 スメクタイ トなどケィ酸化合物では通常、 As a result, in the first liquid mixture, ring respiratory oscillations (approximately 110 cm- 1 ) of 2.5 mM pyridine appeared strongly. On the other hand, no pyridine at this concentration could be observed in the second mixture (normal Raman spectroscopy). Therefore, it was found that the above-mentioned dispersion complex is a substrate for the surface enhancement effect that enables easy analysis using the surface enhancement effect using a commercially available Raman spectrometer. Incidentally, in the case of silicate compounds such as smectite,
485cm"1 Si-O-Si rocking/bend ing of hydrated silicate 485cm " 1 Si-O-Si rocking / bend ing of hydrated silicate
809cm"1 Si02 silicate chain mode 809cm " 1 Si02 silicate chain mode
976cm"1 Si- 0 stretch of bulk chain 976cm " 1 Si-0 stretch of bulk chain
などに Ramanピークが現れるが、第 1混合液ではこれが出現しなかった。 従って、 ケィ酸化合物の千渉がないことも判明した。 この点、 例えば従 来のゾルゲルガラスのラマンスペク トルでケィ酸化合物由来のラマンピ ークがあ らわれてお り 、 ノ ッ ク グラウ ン ド となっ ていた報告 （ F.Raman peaks appeared in the first mixture, but did not appear in the first mixed solution. Therefore, it was also found that there was no interference of the silicate compound. In this regard, for example, Raman peaks derived from silicate compounds appeared in the Raman spectrum of conventional sol-gel glass, and were reported to be a knock ground (F.
Akbarian, B. S. Dunn, and J. I. Zink, J. Raman Spec irosc. , 27 (10) , 775-783 (1996) ： Porous sol-gel silicates containing gold particles as matrixes for surface-enhanced Raman spec t roscopy)と著しく相違 する。 Akbarian, BS Dunn, and JI Zink, J. Raman Spec irosc., 27 (10), 775-783 (1996): significantly different from Porous sol-gel silicates containing gold particles as matrixes for surface-enhanced Raman spec roscopy) I do.
—実施例 2— —Example 2—
上記ゾル調製例の分散複合体 （凝集状態 B) を調製後一日経過した後 に、 その 5 7 0 1 を採り、 0. 0 5 Mのピリジン水溶液 3 0 z l とよ く混合させ、 第 3混合液とした。 同様に、 合成スメクタイ トを含まない 金微粒子凝集液を調製後一日経過した後に、その 5 7 0 a 1 を採り、 0. 0 5 Mのピリジン水溶液 3 0 a 1 とよく混合させ、 第 4混合液とした。 これら 2種の混合液について、 R a m a nモジュールを備えたフ一リエ 変換赤外分光装置 N i c o l e t M a g n a 6 5 0 を用いて、 励起 波長 1 0 6 4 nmでラマンスぺク トルを測定した結果を図 5に示す。 図 5 に見られるように、合成スメク夕イ トを含む第 3混合液では、 2. 5 m Mの低濃度ピリジンの環呼吸振動 （約 1 0 1 0 c m— 1) が強く表れ た。 一方、 合成スメクタイ トを含まない第 4混合液ではこれをほとんど 観察できなかった。 よって、 上記分散複合体は、 1 日経過後も市販のラ マン分光器を用いて簡便に表面増強効果を用いた分析を可能とする、 表 面増強効果の基質であることがわかった。 One day after the preparation of the dispersion complex (aggregation state B) of the above sol preparation example, 5701 was taken and referred to as a 0.05 M aqueous pyridine solution 30 zl. The mixture was mixed well to obtain a third mixed solution. Similarly, one day after the preparation of the aggregated solution of gold microparticles containing no synthetic smectite, 570 a1 was taken and mixed well with a 0.05 M aqueous pyridine solution 30 a1. A mixed solution was obtained. Using a Fourier transform infrared spectrometer Nicolet Magna650 equipped with a Raman module, the Raman spectra of these two mixtures were measured at an excitation wavelength of 1064 nm. See Figure 5. As can be seen in Fig. 5, in the third mixed solution containing synthetic smectite, ring respiratory oscillations (about 110 cm- 1 ) of 2.5 mM low concentration pyridine were strongly exhibited. On the other hand, in the fourth mixed solution containing no synthetic smectite, this was hardly observed. Therefore, it was found that the dispersion complex was a substrate having a surface enhancement effect that allows analysis using the surface enhancement effect easily using a commercially available Raman spectrometer even after one day.
—実施例 3— —Example 3—
上記ゾル調製例の分散複合体 （凝集状態 B ) を室温で保存し、 所定日 数経過後に、 実施例 1の方法でピリジンのラマンスぺク トルを測定し、 環呼吸振動のシグナル強度の推移を、 分散複合体の保存期間に対してプ ロッ トし （図中の翁印）、 図 6 を得た。 対照として実施例 2の第 4混合液 を用いた（図中の X印）。 図 6 に見られるように、 上記分散複合体は、 2 ヶ月近くにわたる長期間、 表面増強効果の基質として働く ことがわかつ た。 一実施例 4一  The dispersion complex (aggregation state B) of the above sol preparation example was stored at room temperature, and after a predetermined number of days, the pyridine Raman spectrum was measured by the method of Example 1, and the transition of the signal intensity of ring respiratory oscillation was measured. Then, the plot was plotted against the storage period of the dispersed complex (Okinaji in the figure), and Fig. 6 was obtained. As a control, the fourth mixed solution of Example 2 was used (marked by X in the figure). As shown in FIG. 6, it was found that the above-mentioned dispersed complex served as a substrate for the surface enhancement effect for a long period of time, which was nearly two months. Example 4
実施例 1 と同様の方法で、 ピリジン水溶液の濃度を変えて、 本発明の 分散複合体を表面増強効果の基質とするピリジン水溶液を分析した。 分 析結果から検量線を作成した結果 図 7に示す。 図 7から本発明による 分散複合体は、 濃度測定に対応できる表面増強効果の基質として働く こ とがわかった。 産業上の利用可能性 In the same manner as in Example 1, the concentration of the pyridine aqueous solution was changed, and the pyridine aqueous solution using the dispersed complex of the present invention as a substrate for the surface enhancement effect was analyzed. Figure 7 shows the result of creating a calibration curve from the analysis results. According to the present invention from FIG. The disperse complex was found to work as a substrate for the surface enhancement effect that can be used for concentration measurement. Industrial applicability
以上のように、 本発明の物質分析方法は、 微量物質あるいは低濃度物 質の分析に有用である。  As described above, the substance analysis method of the present invention is useful for analyzing trace substances or low-concentration substances.

Claims

請求の範囲 The scope of the claims
1 . 群をなして存在し、 表面増強効果を発揮する疎液性微粒子の分散 相中に、 該疎液性微粒子より も分散性の高い微粒子を、 該疎液性微粒子 群の周囲を覆うに十分にたる濃度で共存させてなる分散複合体に、 測定 対象物質を含む流体を接触させ、 光学的計測手段を用いて、 測定対象物 質を分析することを特徴とする物質の分析方法。 1. In the dispersed phase of the lyophobic fine particles present in a group and exhibiting a surface enhancing effect, the fine particles having a higher dispersibility than the lyophobic fine particles are coated around the lyophobic fine particle group. A method for analyzing a substance, comprising contacting a fluid containing a substance to be measured with a dispersion complex coexisting at a sufficient concentration, and analyzing the substance to be measured using an optical measuring means.
2 . 前記疎液性微粒子が凝集剤の作用で群化している請求項 1 に記載 の分析方法。  2. The analysis method according to claim 1, wherein the lyophobic fine particles are grouped by the action of a flocculant.
3 . 光学的計測手段が R a m a n分光法、 赤外分光法の群から選ばれ る請求項 1 に記載の分析方法。 3. The analysis method according to claim 1, wherein the optical measurement means is selected from the group of Ram Spectroscopy and Infrared Spectroscopy.
4 . 疎液性微粒子が、 金、 銀、 銅、 白金、 ニッケル、 イ ンジウム、 パ ラジウムから選ばれる少なく ともひとつ以上の金属を主成分とする請求 項 3に記載の分析方法。  4. The analysis method according to claim 3, wherein the lyophobic fine particles mainly contain at least one metal selected from gold, silver, copper, platinum, nickel, indium, and palladium.
5 . 疎液性微粒子より も分散性の高い微粒子が合成スメク夕イ トなど の膨潤性層状ケィ酸塩の群から選ばれる請求項 4に記載の物質の分析方 法。 5. The method according to claim 4, wherein the fine particles having higher dispersibility than the lyophobic fine particles are selected from the group of swellable layered silicates such as synthetic smectite.
6 . 分散複合体の性状が水を主たる媒質としたゾルであり、 該疎液性 微粒子群の周囲を覆うに十分にたる分散性の高い微粒子濃度が 0 . 1 g Z L以上である請求項 1 に記載の分析方法。  6. The dispersion complex is a sol containing water as a main medium, and the concentration of fine particles having a high dispersibility sufficient to cover the periphery of the lyophobic fine particles is 0.1 g ZL or more. Analysis method described in 1.
7 . 分散複合体の性状が水を主たる媒質としたゾルであり、 該疎液性 微粒子群の周囲を覆うに十分にたる分散性の高い微粒子濃度が 3 0 0 g Z L以下である請求項 1 に記載の分析方法。  7. The property of the dispersion complex is a sol using water as a main medium, and the concentration of fine particles having a high dispersibility sufficient to cover the periphery of the lyophobic fine particles is 300 g ZL or less. Analysis method described in 1.
8 . 分散複合体の性状が水を主たる媒質としたゾルから得られたゲル である請求項 1に記載の分析方法。  8. The analysis method according to claim 1, wherein the property of the dispersion complex is a gel obtained from a sol containing water as a main medium.
9 . 該分散複合体に、 測定対象物質を含む流体と接触させ、 測定対象 物質を分析する系が流れ系である請求項 1 に記載の分析方法。 9. Contact the dispersion complex with the fluid containing the substance to be measured The analysis method according to claim 1, wherein the system for analyzing the substance is a flow system.
1 0 . 膨潤性層状ケィ酸塩が、 配位子化合物、 抗体、 抗原、 酵素、 酵 素基質、 核酸、 核酸の補体からなる群から選ばれるひとつ以上の物質を 修飾したことを特徴とする請求項 5に記載の分析方法。  10. The swellable layered silicate is characterized by modifying at least one substance selected from the group consisting of a ligand compound, an antibody, an antigen, an enzyme, an enzyme substrate, a nucleic acid, and a complement of a nucleic acid. The analysis method according to claim 5.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007049487A1 (en) * 2005-10-25 2007-05-03 Kyushu University, National University Corporation Substrate for analysis for use in raman spectroscopic analysis and substrate assembly for analysis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060275911A1 (en) * 2005-06-03 2006-12-07 Shih-Yuan Wang Method and apparatus for moleclular analysis using nanostructure-enhanced Raman spectroscopy
CN101987364B (en) * 2010-09-14 2012-06-20 江南大学 Method for preparing functional gold nanoparticles with high stability
JPWO2015133408A1 (en) * 2014-03-01 2017-06-15 福岡 隆夫 Nano-beacon and anti-counterfeiting technology using it
JP6499846B2 (en) * 2014-11-17 2019-04-10 株式会社堀場製作所 Absorption spectrometer and calibration curve creation method thereof
WO2020171010A1 (en) 2019-02-20 2020-08-27 隆夫 福岡 Method for representing numerical information

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1161209A (en) * 1997-08-15 1999-03-05 Kdk Corp Dispersion of fine noble metal particles, its production, structure device utilizing the same and its production

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3793786B2 (en) 1995-09-04 2006-07-05 アークレイ株式会社 Method for preventing colloidal aggregation
US6149868A (en) * 1997-10-28 2000-11-21 The Penn State Research Foundation Surface enhanced raman scattering from metal nanoparticle-analyte-noble metal substrate sandwiches
JP2003504642A (en) * 1999-07-16 2003-02-04 ダブリューエム・マーシュ・ライス・ユニバーシティー Metallic fine shell for biosensing applications

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1161209A (en) * 1997-08-15 1999-03-05 Kdk Corp Dispersion of fine noble metal particles, its production, structure device utilizing the same and its production

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAKAMURA KEITARO ET AL.: "Kin. Nendo kyouzon gel tai no chousei to hyomen zoukyou raman sanran tokusei", FUNTAI KOUGAKKAI, 2000 NENDO SHUUKI KENKYU HAPPYOUKAI KOUEN RONBUNSHUU, 2000, pages 44 - 46, XP002955586 *
See also references of EP1376098A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007049487A1 (en) * 2005-10-25 2007-05-03 Kyushu University, National University Corporation Substrate for analysis for use in raman spectroscopic analysis and substrate assembly for analysis

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